My immediate goal is to acquire the necessary skills and expertise to make a successful transition to independence in the academic field of basic cell and cancer biology. The long-term goals of this work are to advance our knowledge on the molecular mechanisms that regulate the interaction of K-Ras with the plasma membrane (PM). The Ras GTPases comprising three main isoforms H-, N- and K-Ras operate at the PM as molecular switches in essential signaling pathways. Oncogenic mutant K-Ras is highly prevalent in multiple human tumors. Despite significant efforts to directly target Ras activity, no anti-Ras drugs have been developed and taken into the clinic. Since Ras proteins must be anchored to the inner leaflet of the PM for full biological activity, I propose that inhibition of K-Ras PM interaction is a valid therapeutic approach to abrogate oncogenic K-Ras activity. Recently, we performed a high content cell-based screen (HCS) of chemical and human siRNA libraries to identify both exogenous and endogenous regulators of the K-Ras PM interaction. We identified three classes of compounds and a set of proteins that induce K-Ras dissociation from the PM. The mechanisms, which reduced K-Ras signaling were: 1) increased K-Ras phosphorylation by AMPK/eNOS/PKG pathway, and 2) perturbation of cellular phosphatidylserine (PS) distribution. Specific Aim 1 will characterize cGMP-activated protein kinase (PKG) as a novel K-Ras kinase regulated by the AMPK/eNOS/PKG pathway. Specific Aim 2 will develop novel strategies to abrogate K-Ras PM binding and signal output by reducing PS PM levels and inducing K-Ras phosphorylation. Specific Aim 3 will characterize a set of proteins that regulate cellular PS distribution. These aims will be addressed by combining my experience in molecular and cell biology to the techniques available in the mentor's laboratory, and will set the path to future investigations aiming to the abrogation of oncogenic K-Ras signal output. Collectively, the overall theme of this proposal is to characterize these novel mechanisms, whereby K-Ras interaction with the PM can be regulated, and be a tractable therapeutic approach to target K-Ras-driven cancer cells.